Novel monofunctional polyethylene glycol aldehydes

ABSTRACT

The present invention provides novel monofunctional polyethylene glycol aldehydes for the pegylation of therapeutically active proteins. The pegylated protein conjugates that are produced, retain a substantial portion of their therapeutic activity and are less immunogenic than the protein from which the conjugate is derived. New syntheses for preparing such aldehydes are described.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The priority of Korean Application No. 10-2001-0078244, filedDec. 11, 2001, as well as U.S. Ser. No. 60/348,452, filed Jan. 6, 2002,U.S. Ser. No. 60/381,503, filed May 17, 2002, U.S. Ser. No. 60/407,741,filed Sep. 3, 2002, and U.S. Ser. No. 10/303,260, filed Nov. 25, 2002are claimed. This Application is a continuation in part of Ser. No.10/303,260, filed Nov. 25, 2002.

BACKGROUND

[0002] Therapeutic proteins which are generally administered byintravenous injection may be immunogenic, relatively water insoluble,and may have a short in vivo half-life. The pharmacokinetics of theparticular protein will govern both the efficacy and duration of effectof the drug. It has become of major importance to reduce the rate ofclearance of the protein so that prolonged action can be achieved. Thismay be accomplished by avoiding or inhibiting glomerular filtrationwhich can be effected both by the charge on the protein and itsmolecular size (Brenner et al., (1978) Am.J.Physiol.,234,F455). Byincreasing the molecular volume and by masking potential epitope sites,modification of a therapeutic polypeptide with a polymer such aspolyethylene glycol (PEG) has been shown to be efficacious in reducingboth the rate of clearance as well as the antigenicity of the protein.Reduced proteolysis, increased water solubility, reduced renalclearance, and steric hindrance to receptor-mediated clearance are anumber of mechanisms by which the attachment of a PEG polymer to thebackbone of a polypeptide may prove beneficial in enhancing thepharmacokinetic properties of the drug. Thus insulin to produce a lessimmunogenic product while retaining a substantial proportion of thebiological activity.

[0003] PEG modification requires activation of the PEG polymer that isaccomplished by the introduction of an electrophilic center. The PEGreagent is now susceptible to nucleophilic attack, predominantly by thenucleophilic epsilon-amino group of a lysyl residue. Because of thenumber of surface lysines present in most proteins, the PEGylationprocess can result in random attachments leading to mixtures which aredifficult to purify and which may not be desirable for pharmaceuticaluse.

[0004] There are a large variety of PEG reagents that have beendeveloped for the modification of proteins. This involves the covalentattachment of a PEG molecule via the formation of a linking groupbetween the PEG polymer and the protein (see for example Zalipsky, etal., and Harris et. al., in: Poly(ethylene glycol) Chemistry:Biotechnical and Biomedical Applications; (J. M. Harris ed.) PlenumPress: New York, 1992; Chap.21 and 22). Some of these reagents are, tovarious degrees, unstable in the aqueous medium in which the PEGylationreaction occurs. In addition, the conjugation process often results inthe loss of in vitro biological activity that is due to several factorsforemost of which being a steric interaction with the proteins activesites. A desired property therefore of a new reagent would be one thatis not susceptible to degradation in an aqueous medium and one that maybe employed to affect the site specific modification of a protein. A PEGaldehyde may be considered such a reagent. For site specific N-terminalpegylation see Pepinsky et al., (2001) JPET,297,1059 (Interferon-β-1a)and U.S. Pat. No. 5,824,784(1998) to Kinstler et al., (G-CSF). The useof a PEG-aldehyde for the reductive amination of a protein utilizingother available nucleophilic amino groups, is described in U.S. Pat. No.4,002,531(1977) to Royer, in EP O 154 316, by Wieder et al., (1979)J.Biol.Chem. 254,12579, and Chamow et al., (1994) Bioconjugate Chem. 5,133.

SUMMARY OF INVENTION

[0005] In accordance with this invention, it has been discovered thataldehydes of the formula:

[0006] IA

[0007] wherein R is hydrogen or lower alkyl, X and Y are individuallyselected from —O— or —NH— with the proviso that X is NH when m is 1 andY is —O—, PAG is a divalent residue of polyalkylene glycol resultingfrom removal of the terminal hydroxy groups, having a molecular weightof from 1,000 to 100,000 Daltons, z is an integer of from 2 to 4, m isan integer of from 0 to 1, and w is an integer of from 2 to 8.

[0008] IB

[0009] wherein A is a polyethylene glycol residue with its two terminalhydroxy groups being removed having a molecular weight of from 1,000 to100,000 Daltons and having a valence of from 1 to 5, n is an integer offrom 1 to 5 which integer is the same as the valence of A, R and w areas above.

[0010] IC

[0011] wherein PAG¹ and PAG² are independently divalent residues of polylower alkylene glycol resulting from removal of the two terminal hydroxygroups with the PAG¹ and PAG² residues having a combined molecularweight of from 1,000 to 100,000 Daltons, R and R¹ are individually loweralkyl or hydrogen, p is an integer of from 1 to 5, and z and w are asabove.

[0012] are useful for conjugation to therapeutically active proteins toproduce PAG protein conjugates which retain a substantial portion oftheir therapeutic activity and are less immunogenic than the proteinfrom which the conjugate is derived.

[0013] In accordance with this invention, a new synthesis has been foundfor the aldehyde of formula:

RO—PAG—O(CH₂)_(z)—O—(CH₂)_(w)—CHO  ID

[0014] wherein R, PAG, z and w are as above.

[0015] which like the compounds of formula IA, IB and IC, is a reagentfor producing PAG protein conjugates.

DETAILED DESCRIPTION

[0016] The aldehyde reagents of formula IA, IB, IC and ID can beconjugated to therapeutically active proteins to produce therapeuticallyactive protein conjugates which retain a substantial portion of thebiological activity of the protein from which they are derived. Inaddition, the reagents of this invention are not susceptible todegradation in the aqueous medium in which the pegylation reaction iscarried out. Furthermore, the aldehyde reagents of this invention can beconjugated to the protein in a controlled manner at the N-terminus. Inthis way, these aldehydes produce the desired conjugates and avoidrandom attachment leading to mixtures that are difficult to purify andwhich may not be desirable for pharmaceutical use. This is extremelyadvantageous since not only are the purification procedures expensiveand time consuming but they may cause the protein to be denatured andthus bring about an irreversible change in the proteins tertiarystructure.

[0017] The therapeutic proteins which can be conjugated in accordancewith this invention can be any of the conventional therapeutic proteins.Among the preferred proteins are included interferon-alpha,interferon-beta, consensus interferon, G-CSF, GM-CSF, EPO, hemoglobin,interleukins, colony stimulating factor, as well as immunoglobulins suchas IgG, IgE, IgM, IgA, IgD and fragments thereof.

[0018] The term polyalkylene glycol designates poly(loweralkylene)glycol radicals where the alkylene radical is a straight orbranched chain radical containing from 2 to 7 carbon atoms. The term“lower alkylene” designates a straight or branched chain divalentalkylene radical containing from 2 to 7 carbon atoms such aspolyethylene, polypropylene, poly n-butylene, and polyisobutylene aswell as polyalkylene glycols formed from mixed alkylene glycols such aspolymers containing a mixture of polyethylene and polypropylene radicalsand polymers containing a mixture of polyisopropylene, polyethylene andpolyisobutylene radicals. The branched chain alkylene glycol radicalsprovide the lower alkyl groups in the polymer chain of from 2 to 4carbon atoms depending on the number of carbon atoms contained in thestraight chain of the alkylene group so that the total number of carbonsatoms of any alkylene moiety which makes up the polyalkylene glycolsubstituent is from 2 to 7. The term “lower alkyl” includes lower alkylgroups containing from 1 to 7 carbon atoms, preferably from 1 to 4carbon atoms, such as methyl, ethyl, propyl, isopropyl, etc. with methylbeing especially preferred.

[0019] In accordance with a preferred embodiment of this invention, PAGin the compound in formulas IA, IC and ID is a polyethylene glycolresidue formed by removal of the two terminal hydroxy groups. Further inaccordance with this invention, PAG in the compound of formula IA, ICand ID, and the A in the compound of formula IB, have molecular weightsof from about 10,000 to 50,000 most preferably from about 20,000 toabout 40,000. In the compound of formula IC it is generally preferredthat the radicals PAG¹ and PAG² have a combined molecular weight of fromabout 10,000 to 50,000 and most preferably from about 20,000 to 40,000.In the compound of formula IC it is generally preferred that p be aninteger of from 1 to 5.

[0020] The aldehydes of compounds of formula IA, IB, IC and ID are usedin forming polyalkyleneoxy protein conjugates. The aldehydes of thisinvention are intermediates for conjugation with the terminal aminogroup as well as other free amino groups on the protein to produce atherapeutically effective conjugate that has the therapeutic propertiesof the native protein. These conjugates when compared to the proteinsfrom which they are derived, show a reduced rate of clearance, decreasedantigenicity, diminished in vivo proteolysis, increased watersolubility, reduced renal clearance, and less susceptibility toreceptor-mediated clearance. All of these factors can help make theconjugate a more effective therapeutic agent than the unmodified proteinitself. The aldehydes of this invention are converted to their proteinconjugates in accordance with the following reaction scheme:

[0021] wherein PNH₂ is a protein covalently attached to a PEG via anucleophilic amino group of the protein.

[0022] In this reaction scheme, G—CHO in the compound of formula V is acomposite of the compounds of IA, IB, IC and ID showing the reactivealdehyde group. In the compound of formula IB, the number of aldehydegroups are in accordance with the valence “n”. If “n” were 4, thereaction in this scheme will take place at four different sites in thiscompound of formula V. In this reaction scheme, P is the proteincontaining a nucleophilic —NH2 group that is conjugated with thecompounds of formula IA, IB, IC and ID.

[0023] As is illustrated in the above reaction scheme, there is anequilibrium that is established between the aldehyde V and itsconventional hydrate IV. This equilibrium is pH dependent, andconsequently the concentrations found for the aldehyde V and its hydrateIV, will depend largely on the particular acidity of the solution. Thepolyalkylene aldehyde of formula V, is reacted with the amine of theprotein to form the imine linkage of formula VI. This imine linkage ofthe compound of formula VI is then reduced to an amine through the useof reducing agents such as cyanoborohydride to give the saturatedconjugated protein of formula VII. The reaction whereby aldehydes areconjugated with proteins through reductive amination is set forth inU.S. Pat. No. 4,002,531, EPO 154,316 and U.S. Pat. No. 5,824,784.

[0024] In reacting the compound of formula V with P—NH₂, one can controlthis reaction so that the aldehydes of formula IA, IB, IC and ID onlyreact at a single site located at the N-terminus amine on the protein.This can be done by carrying out the reaction of the compound of formulaV with P—NH₂ at a pH of from 5.5 to 7.5. In carrying out this reaction,various buffers which maintain the reaction media at a pH of from 5.5 to7.5 can be used. If one wants the amination to proceed on more than oneamino site on the protein, then one carries out the reaction at a pH of8.0 and above, preferably at a pH of from 8 to 10. In this manner, aminogroups, as well as, the N-terminal amino group on the protein areaminated with the PAG aldehydes of this invention.

[0025] The specific PEGylating reagents of formula IA, IB, IC and ID ofthis invention, are stable in aqueous medium and not subject to aldoldecompositions under the conditions of the reductive amination reaction.The amino groups on proteins such as those on the lysine residues arethe predominate nucleophilic centers for the condensation of thealdehydes of this invention. However by controlling the pH of thereaction one can produce a site specific introduction of a polyalkyleneglycol polymer on the protein at the desired N-terminus amino acid. Whenthe compounds of formula IA are conjugated to a protein as is shown inScheme 1, the resulting compound is:

[0026] wherein R, P, Y, PAG, X, m, w and z are as above.

[0027] When the compound in formula IB is conjugated to a protein as isshown in Scheme 1, the resulting compound is:

[0028] wherein A,P, n and w are as above.

[0029] When the compound of formula IC is conjugated to a protein as isshown in Scheme 1, the resulting compound is:

[0030] wherein R, R¹, P, PAG¹, PAG², p, w and z are as above.

[0031] When the compound of formula ID is conjugated with the protein asis shown in Scheme 1 the resulting compound is:

RO—PAG—O—(CH₂)_(z)—O(CH₂)_(w)CH₂NHP  III-D

[0032] wherein R, PAG, P, z and w are as above.

[0033] In accordance with this invention, in formula I A, when m is 0and X is —NH—, these compounds have the formula:

[0034] wherein R, PAG, z and w are as above.

[0035] The compound of formula I-Ai can be prepared by the followingreaction scheme:

[0036] wherein R, PAG, z and w are as above, R₂ is lower alkyl, and OLis a leaving group.

[0037] In the first step of the reaction to produce the compound offormula I-Ai, the acid group of the compound in formula VIII isactivated to produce the compound of formula IX. This is accomplished byactivating the acid group on the compound of formula VIII with anactivating agent to produce a leaving group such as an N-hydroxysuccinimide group. Any conventional method of converting a carboxy groupinto an activating leaving group such as an N-hydroxy succinimide groupcan be utilized to produce the compound of formula IX. In the next stepof the synthesis, the compound of formula IX containing the activatingleaving group is reacted with the amine acetal compound of formula X toproduce the compound of formula XI. This reaction to form the amide offormula XI is carried out by any conventional means of condensing anamine with an activated carboxylic acid group. The compound of formulaXI has the aldehyde protected as its acetal, preferably a lower alkylacetal. Any conventional aldehyde protecting groups such as other alkylacetals can also be utilized. The acetal of formula XI can be hydrolyzedto form the corresponding aldehyde of formula I-Ai. Any conventionalmeans of hydrolyzing an acetal to form the corresponding aldehyde can beutilized to convert the compound of formula XI into the correspondingaldehyde of formula I-Ai.

[0038] In accordance with an other embodiment of preparing a compound ofthe formula I-Ai, the acetal of formula X is replaced with a dioxolaneof the formula Xa that is reacted with compound IX to produce theintermediate of XIa. Compound XIa is then hydrolyzed to thecorresponding vicinal diol and then oxidized with periodate to producethe aldehyde of formula I-Ai.

[0039] wherein R, PAG, z and w are as above, OL is a leaving group, andR₁₃ is hydrogen, alkyl, or phenyl.

[0040] In the compound of formula IA where m is 1, X is —NH— and Y is—O—, this compound has the formula:

[0041] wherein R, PAG, z and w are as above.

[0042] The compound of formula I-Aii can be prepared by the followingreaction scheme.

[0043] wherein OL, R, R₂, PAG, z and w are as above.

[0044] In the above reaction scheme the compound of formula XII is firstreacted with a compound of formula XIII which is a halo formatecontaining a leaving group. Any conventional leaving group can beutilized as OL such as the leaving groups herein before mentioned. Thepreferred leaving group is a para-nitro phenol radical. One can utilizeany of the conventional conditions for reacting an alcohol such as thecompound of formula XII with a chloro formate such as the compound offormula XIII to produce the carbonate of formula XIV. The carbonate isthen reacted with the amine of formula X to produce the compound offormula XV. This reaction is carried out as described hereinbefore withregard to reacting the compound of formula IX with the compound offormula X. The compound of formula XV is then hydrolyzed to produce thecompound of formula I-Aii in the conventional manner as described inconnection with the hydrolysis of the compound of formula XIhereinbefore.

[0045] In the same manner described in the alternate synthesis of I-Ai,the compound of formula Xa is reacted with the carbonate XIV to producethe compound of formula XVa which is then hydrolyzed and oxidized togive the aldehyde I-Aii.

[0046] I-Aii

[0047] wherein R, PAG, z and w are as above, OL is a leaving group, andR₁₃ is hydrogen, alkyl, or phenyl.

[0048] In accordance with another embodiment of this invention whereinthe compound of formula IA, m is 1 and Y and X are both —NH—, thiscompound has the formula

[0049] wherein R, PAG, z and w are as above.

[0050] The compound of formula I-Aiii can be produced by the followingreaction scheme.

[0051] wherein R, PAG, z and w are as above and R₂ is lower alkyl.

[0052] In accordance with this embodiment, the compound of formula XVIis condensed with the compound of formula XVII in a halogenatedhydrocarbon solvent to produce the compound of formula XVIII. Thisreaction utilizes conventional condensing procedures commonly used inreactions between an activated carbonate and an amine. The compound offormula XVIII is condensed with the amine of formula X in an inertorganic solvent to produce the acetal of formula XIX. Any conventionalinert organic solvent can be used in this reaction. The acetal offormula XIX is then hydrolyzed in acidic medium, in the manner describedhereinabove to produce the compound of formula I-Aiii.

[0053] In the same manner described in the alternate synthesis of I-Ai,the compound of formula Xa is reacted with the carbamate XVIII toproduce the compound of formula XIXa which is then hydrolyzed andoxidized to give the aldehyde I-Aiii.

[0054] I-Aiii

[0055] wherein R, PAG, z and w are as above, OL is a leaving group, andR₁₃ is hydrogen, alkyl, or phenyl.

[0056] The preparation of the compounds of the type Xa where R₁₃ ishydrogen has been described for w=2 (Petrov et al., Zh. Obshch. Khim.1962, 32, 3720), w=3 (Olsen et al., J. Org. Chem. 1985, 50, 896) and w=4(Timofeev et al., Nucleic Acids Res. 1996, 24, 3142).

[0057] In the compound of formula IA where m is 1, Y is —NH— and X is—O— the compound has the following formula:

RO—PAG—O(CH₂)_(z)—NHCOO(CH₂)_(w)CHO  I-Aiv

[0058] wherein R, PAG, z and w are as above.

[0059] The compound of formula I-Aiv is prepared by means of thefollowing reaction scheme:

[0060] wherein R,R₁₃, PAG, z and w are as above.

[0061] In this reaction, the starting material of formula XX is atri-hydroxy compound having a terminal primary hydroxy group which isseparated by at least two carbon atoms from two other hydroxy groupsthat are vicinal to each other. The compound of formula XX is convertedto its acetonide derivative of formula XXI by reacting the two vicinalhydroxy groups with acetone leaving free the third hydroxy group. Anyconventional method of forming an acetonide derivative from the twovicinal hydroxy groups can be utilized to carry out this reaction toform the compound of formula XXI. Reagents other than acetone, which areknown to form cyclic acetals with 1,2-diols, may also be used. The freehydroxy group in the acetonide derivative of formula XXI is thenactivated with an activating group such as the p-nitro phenyl chloroformate as is shown in the reaction scheme. This reaction to convert thehydroxy group into an activated derivative is well known in the art. Inthis manner the compound of formula XXII is produced where the primaryhydroxy group on the compound of formula XXI is activated. The compoundof formula XXII is then condensed with the PEG amine of formula XVI toform the condensation product of formula XXIII. Any conditionsconventional in reacting an activated alcohol with an amine to produce aurethane can be utilized to carry out this condensation. The compound offormula XXIII containing the acetonide is then cleaved utilizingconditions conventional in cleaving acetonides such as by treatment witha mild acid, to produce the corresponding di-hydroxy compound. Theresulting dihydroxy groups are then oxidized with mild oxidizing agentssuch as a periodate oxidizing agent to produce the aldehyde of formulaI-Aiv. Any conventional method of oxidizing a vicinal di-hydroxycompound to the corresponding aldehyde can be utilized to carry out thisconversion.

[0062] The compound of formula IB is synthesized from RO—PEG—OR byreaction with acrylic acid by the following reaction scheme:

[0063] wherein A, R, w, n and R₂ are as above.

[0064] The acrylic acid of formula XXV can be reacted with thepolyethylene glycol polymer of formula XXIV in the manner disclosed inU.S. Pat. No. 4,528,334 to Knopf, et al., to produce the compound offormula XXVI. The addition of acrylic acid across the variouspolyethylene glycol units in the series of polyethylene glycol residuesdesignated A can be controlled so that from 1 to 5 bonds with theacrylic acid will take place to form the acrylic acid graft copolymer offormula XXVI. In this manner depending upon the conditions used, asdisclosed in U.S. Pat. No. 4,528,334, from 1 to 5 additions of acrylicacid will occur in the polyethyleneoxy chain. In accordance with thisinvention, an activated form of the carboxy group of the graft copolymerof formula XXVI is reacted with the compound of formula X to form thecompound of formula XXVII via amide formation. This reaction is carriedout in the same manner as described hereinbefore in connection with theconversion of the compound of formula VIII to the compound of formula XIby reaction of the compound of formula X, through the use of anappropriate carboxy activating leaving group as in formula IX. By theprocedure described in connection with the conversion of the acetal ofXI to the aldehyde of formula I-Ai, the acetal of formula XXVII wasconverted to the derivative IB.

[0065] The compound of formula IC can be prepared as shown by thefollowing reaction scheme:

[0066] wherein R, R₂, R¹, PAG¹, PAG², OL, p, w and z are as above.

[0067] The derivative of formula IC is prepared from a compound offormula XXVIII in which there is an activated carboxyl group. Thiscarboxyl group can be activated in the manner disclosed hereinbeforewith respect to the activation of the compound of the formula VIII toproduce the compound of the formula IX. The activated compound is thencondensed with the amino acetal compound of formula X to produce thecompound of formula XXIX in the same manner as described hereinbefore inconnection with the reaction of the compound of formula IX with thecompound of formula X to produce the compound of formula XI. Thecompound of formula XXIX is next converted to the compound of theformula IC by acid hydrolysis as described herein before in connectionwith the preparation of the compound of formula I-Ai from compound XI.

[0068] In the same manner described in the alternate synthesis of I-Ai,the compound of formula Xa is reacted with the derivative XXVIII toproduce the compound of formula XXIXa which is then hydrolyzed andoxidized to give the aldehyde IC.

[0069] wherein R, R¹, PAG¹, PAG², R₁₃, OL, p, w and z are as above.

[0070] The compound of formula ID is produced from a compound of theformula XII via the following reaction scheme:

[0071] wherein R,R₁₃,PAG, and w and z are as above, X may be a halogenor sulfonate ester and B is an alkalai metal.

[0072] In carrying out this process the compound of formula XII isconverted to the compound of formula XXX by converting the hydroxy groupon the compound of formula XII to an activating leaving group. Theconversion of the terminal hydroxyl group of compound XII into anactivated halide leaving group X, can be readily achieved by reactionwith a conventional halogenating reagent such as thionyl bromide. On theother hand where leaving groups, other than halides, are utilized, thehydroxy group of the compound of formula XII may be converted to asulfonate ester by reaction with a halide of the activating leavinggroup such as mesyl or tosyl chloride. Any conventional method forconverting the hydroxy group of compound XII to an activating leavinggroup such as a tosylate or mesylate or any of the aforementionedleaving groups can be utilized to produce the compound of formula XXX.This reaction may be carried out by reacting the formula XII with ahalide of an activating leaving group such as tosyl chloride. Thecompound of formula XXX can then be condensed with the alkoxide offormula XXIa to form the compound of formula XXXI. In this case theacetonide group is a precursor to the aldehyde of formula ID. In thecase shown in the above reaction scheme where an acetonide is used, theacetonide can be hydrolyzed in mild acid. However any conventional meansto produce the resulting dihydroxy compound from an acetonide can beused in this conversion. The dihydroxy compound resulting form thishydrolysis can then be oxidized with a periodate to give the aldehyde offormula ID. This aldehyde can be reacted as set forth in Scheme 1 with aprotein at the N-terminal amino acid to form the conjugate of thecompound of formula IIID as described hereinbefore.

[0073] The compound of formula ID can also be produced from a compoundof the formula XXI via the following reaction scheme.

[0074] wherein R, R₁₃, and w are as above, PEG is a divalent residue ofpolyethylene glycol resulting from removal of the terminal hydroxygroups, having a molecular weight of from 1,000 to 100,000 Daltons.

[0075] In this reaction process, the compound of formula XXI is reactedwith any conventional organic alkali metal base such as potassiumnaphthalide to form the corresponding alkoxide XXIa. Liquid ethyleneoxide is then added under conventional polymeric conditions to asolution of XXIa. In this manner the anionic ring opening andpolymerization of ethylene oxide is allowed to proceed under conditionsthat are well known for the production of polyethylene glycol polymers.In addition the amount of polymerization of the ethylene oxide can becontrolled by conventional means to produce a polyethylene polymer ofany desired molecular weight. Any remaining ethylene oxide is thenremoved from the reaction mixture. Reaction for several hours, with anexcess of an alkyl halide such as methyl iodide, results in theformation of a terminal alkyl ether. The product, the compound offormula XXXII, may then be converted to a compound of the formula ID inthe same manner as described hereinbefore for the conversion of thecompound of formula XXXI to the compound of formula ID. The compounds offormula ID and their intermediates, are considered as new derivativeswhen w is an integer from 3 to 8.

[0076] The aldehydes of formula IA, IB, IC and ID can be conjugated asdescribed herein before with various proteins through an amine group onthe protein by the process of reductive amination as disclosed in U.S.Pat. No. 5,824,784 dated Oct. 20, 1998. By means of regulating the pH(i.e. from 5.5 to 7.5) the aldehydes in this invention may condense atthe N-terminus amino group of a protein so as to obtain a monoconjugatederivative. In this manner, the pegylating reagents of IA, IB, IC and IDcan from site specific mono-conjugates with the N-terminal amino groupof various proteins thereby avoiding the necessity of employingextensive purification or separation techniques. On the other hand, ifhigher pH's from about 8.5 and above are utilized, the reductiveamination procedure will also involve the various lysine amino groupswhich are available in the protein molecule. Among the preferredproteins for such conjugations are included G-CSF, GM-CSF, interferon-α,interferon-β, EPO and Hemoglobin.

[0077] In accordance with this invention, when the embodiments offormula I-Ai, I-Aii, I-Aiii and I-Aiv are reacted with the proteins byreaction as shown in Scheme 1, the following compounds are produced.

[0078] wherein P, R, PAG, z and w are as above.

[0079] The following examples are illustrative of the invention and arenot to be construed as limiting the invention. In the followingexamples, the numbering as “1,” etc. refers to the reaction schemefollowing the descriptive portions in each example.

EXAMPLES Example 1 Scheme A (Type I-Ai)

[0080] Synthesis of mPEG-amide-propionaldchyde.

[0081] Methoxy PEG-OH (M.W. 20,000, n=452) 1 and potassium t-butoxidewere dissolved in t-butyl alcohol and stirred at 60° C. Ethylbromoacetate was then slowly added and the mixture stirred for another15 hours at 80-85° C. After filtering the reaction mixture, the solventwas evaporated under reduced pressure. The residue was dissolved indistilled water, washed with diethyl ether, and extracted twice withdichloromethane. The dichloromethane solution was dried over magnesiumsulfate and the solvent removed under vacuum. Precipitation was inducedby the addition of diethyl ether to the crude residue and theprecipitated compound was then filtered and dried under vacuum to givethe product 2 as a white powder.

[0082] The mPEG-ethyl acetate was dissolved in 1 N-sodium hydroxide andstirred for 15 hours at room temperature. The reaction mixture was thenadjusted to pH 2 with 1 N aqueous HCl and extracted twice withdichloromethane. The extracted organic layer was dried over magnesiumsulfate and the organic solvent removed. Diethyl ether was then added tothe residue and the precipitated compound filtered. The product wasdried under vacuum and the resulting acid 3 obtained as a white powder.

[0083] To a solution of the mPEG-acetic acid 3 dissolved indichloromethane and cooled to 0-5° C., was added N-hydroxysuccinimidefollowed by a solution of dicyclohexylcarbodimide in dichloromethane.The reaction mixture was stirred for 15 hours at room temperature. Theby-product, dicyclohexylurea, was removed from the reaction mixture byfiltration and the residual organic solvent evaporated. The cruderesidue was then recrystallized from ethyl acetate filtered, washedtwice with diethyl ether and dried for 12 hours under vacuum to affordthe mPEG-succinimidyl acetate 4 as a white powder (see Example 2).

[0084] The mPEG-succinimidyl acetate 4 was dissolved in dichloromethaneand stirred at room temperature while a solution of 1-amino-3,3-diethoxypropane in dichloromethane was added. The resulting solutionwas then stirred for 2 hours at room temperature. Precipitation wasinduced by the addition of diethyl ether. The product was then filteredand recrystalized from ethyl acetate. The recrystalized compound wasdried under vacuum to give 5 as a white powder.

[0085] The diethyl acetal 5 was dissolved in an aqueous solutioncontaining phosphoric acid (pH1) and stirred for 2 hours at 40-50° C.After cooling the reaction mixture to room temperature, the acidity wasreduced to a pH6 by the addition of a 5% aqueous sodium bicarbonatesolution. Brine was added and the resulting mixture extracted twice withdichloromethane. The organic layer was dried over magnesium sulfate,filtered and the solvent evaporated under reduced pressure.Precipitation was induced by the addition of diethyl ether to the cruderesidue. The product was collected and dried under vacuum to give 6 as awhite powder.

[0086] By using the same procedure, compounds of the type I-Ai can beprepared whereby the integer n may be from 22 to 2,300.

[0087] The integer n may be from 22 to 2,300 but more preferably 22 to1,000.

Example 2 Scheme B (Type I-Ai)

[0088] Synthesis of mPEG-amide-butyraldehyde

[0089] To 10 g, (1 mmol) of polyethylene glycol propionic acid 1, (MW10,000, n=226) dissolved in dry methylene chloride ( 30 ml) was addeddry and finely powdered NHS (0.56 g, 5 mmol). The flask was cooled in anice-water bath and DCC (0.22 g, 1.08 mmol) added. The reaction mixturewas stirred at 0° C. for 1 h and at room temperature for 24 h. Theprecipitated 1,3-dicyclohexylurea (DCU) was removed by filtration, andthe filtrate added to ether (50 ml). After cooling to 4° C. the crudematerial 2 was collected by filtration and purified by precipitatingtwice from methylene chloride by the addition of ether.

[0090] To the N-hydroxy succinate derivative 2 (8.5 g ˜0.85 mmol)dissolved in dry methylene chloride (25 ml) there was added1-amino-4,4-dimethoxybutane ( 0.33 g, 2.5 mmol). The reaction mixturewas stirred at room temperature for 2 h and the product precipitated inether (100 ml). After cooling to 4° C., the crude acetal of formula 3was collected by filtration and then re-dissolved in a minimum ofmethylene chloride. The product was then precipitated by the addition ofether to give 8 g of the acetal as a white solid. The acetal was thendissolved in 50 ml of 0.1M HCL and stirred at room temperature for 4 hto produce the amide aldehyde 4. The water was then removed underreduced pressure, and the crude amide-aldehyde product 4 purified bychromatography. By using the same procedure, compounds of the type I-Aican be prepared whereby the integer n may be from 22 to 2,300.

[0091] The integer n may be from 22 to 2,300 but more preferably 22 to1,000.

Example 3 Scheme C (Type I-Aii)

[0092] Synthesis of mPEG-urethane-propionaldchyde.

[0093] Triphosgene (148 mg, 0.5 mmol) in 5 ml of dichloromethane wasadded slowly to a solution of 10 g of mPEG 1 (0.5mmol) (MW 20,000,n=452)) dissolved in 30 ml of dichloromethane and the resulting mixturestirred for 15 hours at room temperature. The organic solvent was thenremoved under vacuum and the residue washed with dry ether and filtered.The acid chloride was then dissolved in 30 ml of dry dichloromethane andtreated with 80 mg (0.7 mmol) of N-hydroxysuccinimide followed bytriethylamine (71 mg, 0.1 ml). After 3 hours, the solution was filteredand evaporated to dryness. The residue was dissolved in warm (50° C.)ethyl acetate, and then the solution cooled to 0° C. The resultingprecipitate 2 was collected as a white powder, and the product driedunder vacuum.

[0094] To a solution of the 5 g (0.25 mmol) ofmPEG-succinimidylcarbonate 2 dissolved in 30 ml of dichloromethane wasadded 1-amino-3, 3-diethoxypropane (110 mg, 0.75mmol). The reactionmixture was then stirred for 2 hours at room temperature. Ether was thenadded and the resulting precipitate collected and recrystalized fromethyl acetate. The product was washed twice with diethylether and driedunder vacuum to give 3 as a white powder.

[0095] The diethyl acetal 3 (5 g) was dissolved in an aqueous solutioncontaining phosphoric acid (pH1) and stirred for 2 hours at 40-50° C.After cooling the reaction mixture to room temperature, the acidity wasreduced to a pH6 by the addition of a 5% aqueous sodium bicarbonatesolution. Brine was added and the resulting mixture extracted twice withdichloromethane. The organic layer was dried over magnesium sulfate,filtered and the solvent evaporated under reduced pressure.Precipitation was induced by the addition of diethyl ether to the cruderesidue. The product was collected and dried under vacuum to give 4 as awhite powder.

[0096] By using the same procedure, compounds of the type I-Aii can beprepared whereby the integer n may be from 22 to 2,300.

[0097] The integer n may be from 22 to 2,300 but more preferably 22 to1,000.

Example 4 Scheme D (Type I-Aii)

[0098] Synthesis of mPEG-urethane-butyraldehyde.

[0099] To a solution of 201.6 mg (1 mmol) of 4-nitrophenyl chloroformateand 118.6 mg (0.97 mmol) of 4-dimethylaminopyridine dissolved in 10 mlof dry methylene chloride was added dropwise a solution of 9.7 g (0.97mmol) of mPEG 1 (MW10,000, n=225) dissolved in 50 ml of methylenechloride. After stirring for lh at room temperature, 172.8 mg (1.17mmol)of 1-amino-4,4-dimethoxybutane was then added to the solution of the4-nitrophenyl carbonate derivative 2. Stirring was continued for 20 hafter which time the product 3 was precipitated by the addition of ether(100 ml).

[0100] After cooling to 4° C., the crude acetal of formula 3 wascollected by filtration and precipitated twice with ether from amethylene chloride solution to give 8 g of the acetal as a white solid.The acetal was then dissolved in 50 ml of 0.1M HCL and stirred at roomtemperature for 4 h. The water was then removed under reduced pressure,and the crude urethane-aldehyde 4 purified by chromatography.

[0101] By using the same procedure, compounds of the type I-Aii can beprepared whereby the integer n may be from 22 to 2,300.

[0102] The integer n may be from 22 to 2,300 but more preferably 22 to1,000.

Example 5 Scheme E (Type I-Aiii)

[0103] Synthesis of mPEG-urea-propionaldchyde.

[0104] To a solution of 2 g (0.2 mmol) ofalpha-(2-aminoethyl)-omega-methoxypoly(oxy-ethanediyl)(MW 10,000, n=226)of formula 1 in 40 ml of dry methylene chloride, was added at 0° C., 65mg (0.3 mmol) of di-2-pyridyl carbonate 2 and the mixture stirred for 5h. The product of formula 3 was then precipitated by the addition of 100ml of ether, filtered, and washed with an additional 100 ml of ether.The product was then dried under vacuum under a slow stream of nitrogento give 1.9 g of the compound of formula 3 as a white powder. To theresulting urethane intermediate (1.5 g ˜1.5 mmol) dissolved in drymethylene chloride (25 ml) was added 0.6 g (˜4 mmol) of1-amino-3,3-diethoxypropane. The reaction mixture was stirred at roomtemperature for 12 h and the acetal of formula 4 precipitated from ether(100 ml). After cooling to 4° C. the crude acetal was collected byfiltration and precipitated twice from methylene chloride by addition ofether to obtain 1.1 g of the acetal as a white solid. The acetal wasthen dissolved in 50 ml of 0.1M HCL and stirred at room temperature for4 h. The water was then removed under reduced pressure, and the crudeurea-aldehyde product of formula 5 was purified by chromatography (theuse of reagent 3 for urea formation, see U.S. Pat. No. 5,539,063).

[0105] By using the same procedure, compounds of the type I-Aiii can beprepared whereby the integer n may be from 22 to 2,300.

[0106] The integer n may be from 22 to 2,300 but more preferably 22 to1,000.

Example 6 Scheme F (Type I-Aiv)

[0107] Synthesis of mPEG-urethane-butyraldehyde.

[0108] A mixture of pentane-1,2,5-triol 1 (11.7 g, 97.5 mmol) andtoluene-p-sulfonic acid (0.3 g) in acetone-light petroleum ether (bp40-60 ) (1:1 60 ml) was refluxed 24 h with a Dean-Stark apparatus. Thesolvent was then removed under vacuum and the residue dissolved inether. The ethereal solution was washed with aqueous sodium carbonate,dried (Na₂CO₃) and the ether removed. The resulting oil was thendistilled to give 10.7 g of the 1,3-dioxolane-2,2-dimethyl-4-propanol offormula 2 bp. 117-118, 12 mm.(Golding et al., (1978) J. C. S. Perkin II,839).

[0109] To a solution of 11.2 g (55 mmol) of 4-nitrophenyl chloroformatein 100 ml of acetonitrile was added slowly 7.3 g (60 mmol) of4-dimethylaminopyridine followed by 8 g (50 mmol) of the above acetonideproduct 2 dissolved in 20 ml of acetonitrile. After stirring for 24 h,the precipitated pyridinium hydrochloride was filtered and the solventremoved under reduced pressure. The residue was then dissolved in 200 mlof ether and washed with a 5% aqueous solution of sodium bicarbonate.The ether solution was then dried (Na₂CO₃) and the solvent removed undervacuum to give 16 g of the acetonide of formula 3.

[0110] To a solution of 6 g (0.6 mmol) ofalpha-(2-aminoethyl)-omega-methoxypoly(oxy-ethanediyl) (MW 10,000,n=226)of formula 4 in 40 ml of dry methylene chloride, was added at 0° C., 196mg (0.6 mmol) of the 4-nitrophenyl carbonate of formula 3 and 74 mg of4-dimethylaminopyridine. The solution was stirred for 24 h after whichtime the compound of formula 5 was precipitated by the addition of 150ml of ether. This product was filtered and further washed with ether togive 5 g of the urethane-acetonide of formula 5 as a white solid.

[0111] Compound 5 (5 g, 0.5 mmol) was then dissolved in 75 ml of 0.1MHCl and stirred for 6 h. The water and HCl were then removed underreduced pressure to give the corresponding diol product. To 5 g of thediol dissolved in 75 ml water was added 267 mg of NaIO₄ (1.25 mmol) andthe reaction allowed to proceed for 5 h in the dark. The aldehyde offormula 6 was then isolated by size exclusion chromatography on aSephadex G 10 column. Oxidation of the 1,2-diol may also be realizedusing NaIO₄ supported on wet silica gel. Using this procedure thealdehyde is obtained without hydrate formation. (see Vo-Quang et al.,(1989) Synthesis No.1,64).

[0112] By using the same procedure, compounds of the type I-Aiv can beprepared whereby the integer n may be from 22 to 2,300

[0113] The integer n may be from 22 to 2,300 but more preferably 22 to1,000.

Example 7 Scheme G (Type IB)

[0114] Synthesis of Pendant mPEG-urethane-propionaldehyde.

[0115] Nonane was added to a reaction vessel containing mPEG (M.W.20,000), and heated to 140-145° C. When the solid melted, acrylic acidand t-butyl peroxybenzoate (a reaction initiator) were slowly added tothe reaction mixture over a period of 1.5 hours. After the addition, themixture was stirred for an additional hour at 140-145° C. After theremoval of residual nonane from the reaction mixture by evaporation,methanol was added to the mixture and heated and stirred until ahomogeneous solution was obtained. The hot solution was then filteredunder vacuum and the filtrate diluted with a 90/10/, v/v, MeOH/H₂Osolution. The resulting mixture was then filtered through a Pall Filtronultrafiltration system and the filtrate then concentrated under reducedpressure. The residue was dissolved by heating with a 50/50, v/v,acetone/isopropyl alcohol solution, cooled to room temperature, andplaced in the refrigerator overnight. The product 1 was then filtered,washed 3 times with 50/50, v/v, acetone/isopropyl alcohol solution andfinally 3 times with diethyl ether and then vacuum dried overnight. Theacid number of the pendant-PEG-propionic acid 1 was determined bytitration (mg of KOH needed to neutralize one gram of sample).

[0116] The pendant-PEG-propionic acid 1 was dissolved in dichloromethaneand cooled to 0-5° C. N-hydroxysuccinimide was then added followed bythe addition of dicyclohexylcarbodimide dissolved in chloromethane.After stirring for 15 hours at room temperature, the dicyclohexylurea byproduct was removed from the reaction mixture by filtration and theresidual organic solvent evaporated under vacuum (see Example 2). Thecrude residue was recrystalized from ethyl acetate, filtered, washedtwice with diethyl ether, and dried for 12 hours under vacuum to givethe pendant PEG-succinimidyl propionate 2 as a white powder.

[0117] To a solution of the pendant PEG-succinimidyl propionate 2dissolved in dichloromethane was added at room temperature 1-amino-3,3-diethoxypropane and the resulting solution stirred for 2 hours.Precipitation was induced by the addition of diethyl ether and theproduct so obtained recrystalized from ethyl acetate. The recrystalizedcompound was filtered, washed twice with diethyl ether, and dried for 12hours under vacuum to give the pendant-PEG-propoionaldehydediethylacetal 3 as a white powder.

[0118] The pendant-PEG-propionaldehyde diethyl acetal 3 was dissolved inan aqueous solution containing phosphoric acid (pH 1) and stirred for 2hours at 40-50° C. After cooling the reaction mixture to roomtemperature, the acidity was reduced to a pH6 by the addition of a 5%aqueous sodium bicarbonate solution. Brine was added and the resultingmixture extracted twice with dichloromethane. The organic layer wasdried over magnesium sulfate, filtered and the solvent evaporated underreduced pressure. Precipitation was induced by the addition of diethylether to the crude residue. The product was collected and dried undervacuum to give the pendant PEG-amide propionaldehye 4 as a white powder.

[0119] By using the same procedure, compounds of the type IB can beprepared whereby the integer m may be from 22 to 2,300

[0120] The integer m may be from 22 to 2,300 but more preferably 22 to1,000. The integer n may be 1 to 20 and more preferably 1 to 5.

Example 8 Scheme H (Type IC)

[0121] Synthesis of Branched mPEG-amide-propionaldehyde.

[0122] The conversion of the branched chain carboxy acid 1 to thecorresponding propionaldehyde 2 was carried out in the same manner asdescribed in Example 2.

[0123] wherein R, R¹, PAG¹, PAG , p and z are as above.

Example 9 Scheme I (Type IC)

[0124] Synthesis of Branched mPEG-amide-butyraldehyde.

[0125] The conversion of the branched chain carboxy acid 1 to thecorresponding butyraldehyde 2 was carried out in the same manner asdescribed in Example 2.

[0126] wherein R, R¹, PAG¹, PAG², p and z are as above.

Example 10 Scheme J (Type ID)

[0127] Synthesis of mPEG-Butyraldehyde.

[0128] A mixture of pentane-1,2,5-triol of formula 1 (11.7 g, 97.5 mmol)and toluene-p-sulfonic acid (0.3 g) in acetone-light petroleum ether (bp40-60) (1:1 60 ml) was refluxed 24 h with a Dean-Stark apparatus. Thesolvent was then removed under vacuum and the residue dissolved inether. The ethereal solution was then washed with aqueous sodiumcarbonate, dried (Na₂CO₃) and the ether removed. The resulting oil wasthen distilled to give 10.7 g of 1,3 dioxolane-2,2-dimethyl-4-propanolof formula 2 bp. 117-118, 12 mm.(Golding et al., (1978) J. C. S. PerkinII, 839).

[0129] To a solution of 6 g (0.6 mmol) of mPEG alcohol (MW 10,000,n=226) in 40 ml of dry methylene chloride, was added at −10° C., 182 mg(0.26 ml) of trimethylamine and toluene-p-sulfonyl chloride (381 mg, 2mmol). The cooling was removed and the mixture stirred at roomtemperature for 18 h. The product was precipitated by the addition of150 ml of ether, filtered and further washed with ether to give 5 g ofthe PEG tosylate 3 as a white solid.

[0130] A solution of 320 mg (2.0 mmol) of1,3-dioxolane-2,2-dimethyl-4-propanol 2 dissolved in 10 ml of drydioxane was added dropwise under nitrogen to a mixture of 100 mg ofsodium hydride suspended in 5 ml of benzene. The mixture was thenstirred for 30 min to give the sodium alkoxide salt of 2. To thissolution was then added dropwise over a 20-min period, a solution of 4 g(0.4 mmol) of the PEG tosylate 3 dissolved in 30 ml of dioxane. Thereaction mixture was then stirred for 24 h at 40° C. and then addeddropwise to 150 ml of ether to precipitate the compound of formula 4 asa white solid. This material was then purified by chromatography on asmall alumina column.

[0131] The PEG acetonide 4 (3.5 g) was dissolved in 40 ml of 0.1M HCland stirred for 6 h. The water and HCl were then removed under reducedpressure to give the corresponding diol product. To 3 g of the 1,2-dioldissolved in 40 ml water (˜0.3 mmol of diol) was added 160 mg of NaIO₄(0.75 mmol) and the reaction allowed to proceed for 5 h in the dark. ThePEG butyraldehyde 5 was isolated by size exclusion chromatography on aSephadex G 10 column.

[0132] By using the same procedure, compounds of the type ID can beprepared whereby the integer m may be from 22 to 2,300

[0133] The integer n may be from 22 to 2,300 but more preferably 22 to1,000.

What is claimed:
 1. An aldehyde of the formula:RO—PAG—O—(CH₂)_(z)—O—(CH₂)_(w)—CHO wherein R is lower alkyl, PAG is adivalent residue of polyalkylene glycol resulting from removal of theterminal hydroxy groups, having a molecular weight of from 1,000 to100,000 Daltons, z is an integer of from 2 to 4, and w is an integer offrom 3 to
 8. 2. The aldehyde of claim 1 wherein said divalent residue isformed from polyethylene glycol.
 3. The aldehyde of claim 2 wherein theresidue has a molecular weight of 5,000 to 50,000 Daltons.
 4. Thealdehyde of claim 3 wherein R is methyl and said residue has a molecularweight of 20,000 Daltons.